AbdelMaksoud, Rania A.a; Talat, Iman M.b; Khadr, Nouran A.a; Eid, Nagat S.a; ElGarem, Yehya F.a; Hussein, Tarek M.a; Saad, Saad M.c
Psoriasis is one of the most common inflammatory diseases of the skin, characterized by focal formation of inflamed, raised plaques that constantly shed scales derived from excessive growth of skin epithelial cells (Weinstein and Frost, 1968).
It affects ∼2% of the world’s population (Christophers, 2001). Psoriasis was considered to be a T-cell-dependent chronic relapsing inflammatory dermatosis induced by CD8+CD4+ (Th1) T cells that produce type 1 cytokines, such as interferon-γ and tumor necrosis factor-α (Quaglino et al., 2009).
However, the role of Th1 cells in psoriasis is under re-evaluation as a subset of Th17 cells, unique CD4+ T cells characterized by the production of interleukin (IL)-17, has been newly discovered. This subset of cells is located predominantly in the dermis of the lesions (Lowes et al., 2008). However, whether psoriasis is predominantly Th1 mediated or Th17 mediated is still under investigation (Yan et al., 2010).
In general, tolerance is maintained in the periphery through a variety of mechanisms, including active suppression of the function of autoreactive T cells by a population of regulatory T cells (Tregs). These Tregs are identified by their expression of CD4, the IL-2 receptor α-chain (CD25), and the forkhead family transcription factor FOXP3. They can inhibit the development of autoimmunity when transferred into an appropriate host (Fujimura et al., 2008).
Mutation of the human gene FOXP3 results in immunedysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX; Bennett et al., 2001; Wildin et al., 2001). IPEX patients suffer from autoimmune enteropathy, diabetes and thyroiditis, hemolytic anemia, thrombocytopenia, food allergy, and dermatitis (Wildin et al., 2001). Therefore, dermatitis in IPEX can present with features of psoriasis or atopic dermatitis with hyper-IgE (Nieves et al., 2004; Halabi-Tawil et al., 2009). Treg defects are also reported in patients with multiple sclerosis (Viglietta et al., 2004), psoriasis (Sugiyama et al., 2005), and systemic lupus erythematosus (Valencia et al., 2007).
Th1 and Th17 cells and the FOXP3-expressing Tregs can be considered as the yin and yang of the immune system. It is suggested that both Tregs and Th17 cells are differentiated from the same T-cell subset under the influence of transforming growth factor-β, IL-6, and probably because of the effect of other cytokines. During the resting state of the immune system, the production of transforming growth factor-β favors the induction of Tregs, which suppress autoimmunity. However, activation of the innate immune system will lead to the generation of IL-6 and push the balance toward the generation and differentiation of proinflammatory Th17 cells (Bettelli et al., 2007; Karai and Bergfeld, 2009).
Further characterization of Th1, Th17, and Tregs cells can potentially hold the key for the treatment of patients suffering from autoimmune diseases, cancer, or infection, as well as those receiving organ transplants; it may also play a significant role in the prevention and/or treatment of immunity-related diseases in clinics (Karai and Bergfeld, 2009).
Aim of the work
The aim of this study was to examine the presence of CD4+CD25+ FOXP3+ Tregs immunohistochemically in the lesional skin of psoriasis vulgaris and erythrodermic psoriasis, aiming to detect their percentage in the dermis and to correlate the findings with the pathogenesis of the disease.
Patients and methods
Twenty-five patients with psoriasis and 10 normal control participants were recruited for skin biopsy. All patients were subjected to detailed history taking, clinical examination, and Psoriasis Area and Severity Index (PASI) scoring, as well as skin biopsy for hematoxylin and eosin (H&E) conventional staining and FOXP3 immunostaining. Of these 25 patients, 17 patients had psoriasis vulgaris (the PASI score ranged from 5 to 40), whereas eight had erythrodermic psoriasis (the PASI score ranged from 60 to 70). Of the 17 patients with psoriasis vulgaris, four patients had generalized psoriasis, whereas 13 patients had localized psoriasis. Biopsy specimens involved the center, border, and perilesional zone, respectively. The control specimens were obtained from the Plastic Surgery Department, Alexandria University, from residual specimens discarded after plastic surgery of the abdomen; they were formalin fixed and paraffin-embedded.
The patients enrolled in the study had not used any local therapy for 2 weeks and had not received any systemic treatment for 4 weeks before the skin biopsy was taken. Informed consent was obtained before commencing the study. The study protocol was approved by the Ethics Committee, Faculty of Medicine, Alexandria University.
Patients with a history of the following diseases were excluded from the study:
Type I diabetes mellitus.
Systemic lupus erythematosus.
Inflammatory bowel disease.
Psoriatic area and severity index (PASI score; Harari et al., 2000)
PASI was originally described as a method for quantifying the extent of the disease and for evaluating its improvement with treatment. It is the gold standard to assess psoriasis, although it is time-consuming and may take 10–15 min even for experienced personnel.
The basis for obtaining the PASI score is the evaluation of four separate body areas, head, trunk, and upper and lower extremities, after scoring them separately for erythema, infiltration, and desquamation, after establishing the extent of skin surface involved, to obtain a final value between 0 and 72.
The PASI score is calculated as follows:
where E=erythema; I=infiltration; D=desquamation; A=area; h=head; t=trunk; u=upper extremities; and l=lower extremities.
A numerical value is assigned to the extent of the lesions in each area (area score):
1=<10%, 2=10±30%, 3=30±50%, 4=50±70%, 5=70±90%, and 6=90±100%.
E, I, and D are assessed according to a five-point scale: 0=no symptoms, 1=slight, 2=moderate, 3=marked, 4=very marked.
Five-micrometer-thick sections cut from paraffin blocks were H&E stained and examined microscopically to confirm the diagnosis and assess the intensity of staining of the mononuclear dermal cellular infiltrate.
Unstained slides of formalin-fixed, paraffin-embedded tumor specimens were used for immunohistochemical procedures. Representative samples were stained using the monoclonal antibodies CD3, CD4, and CD8 to identify the subset of the mononuclear infiltrate in the dermis. All the specimens included were stained using the primary monoclonal antibody FOXP3, used at a concentration of 10 µg/ml. Positive controls (normal tonsil) and negative controls were included in all the runs. Heat-mediated antigen retrieval using a microwave was performed before commencing the immunohistochemical staining protocol. The streptavidin–biotin–peroxidase complex method was used. This technique involves sequential incubation of the specimens with an unconjugated primary antibody specific to the target antigen, a biotylinated secondary antibody that reacts with the primary antibody, enzyme-labeled streptavidin, and diaminobenzidine substrate chromogen. The detection kit Ultra Vision Detection System (antipolyvalent, ready to use, HRP/diaminobenzidine; Lab Vision Corporation, Fremont, California, USA) was used.
Interpretation of immunohistochemical results
For each skin biopsy specimen, the overall percentages of FOXP3 cells with respect to the skin-infiltrating lymphocytes were calculated from three individual values obtained from three consecutive microscopic high-power fields of the immune-stained sections (magnification ×200).
For the quantitative assessment of the degree of H&E staining or FOXP3 immune staining of the dermal mononuclear inflammatory cellular infiltrate, the following scale was proposed: negative: no cells detected, 0–1: very minimal expression, 1+: mild expression, 2+: moderate expression, 3+: dense expression.
Image cytometric analysis
A photomicrograph of each microscopic slide was captured at a magnification of ×400 using a digital video camera (C5060; Olympus, Tokyo, Japan) mounted on a research light microscope (BX60; Olympus). Images were then transferred to the computer system for analysis.
All the steps performed for the evaluation of immunopositivity were carried out using image analysis software (Image J, 1.41a; NIH, Hercules, California, USA). Phase analysis was carried out automatically to determine the count of immunopositive cells, the total surface area of immunopositivity, as well as area fraction (ratio of the area of immunopositive cells to the total area of the microscopic field).
The data collected were tabulated using Microsoft Excel (Microsoft Office 2007, Microsoft, Redmond, Washington, USA).
Statistical analyses were carried out using SPSS Statistics 20. Qualitative data were described as number and percentage. Quantitative ordinal data were described using median, minimum, and maximum. Correlations between different quantitative variables were tested using Spearman’s correlation test. The Mann–Whitney U-test was used to compare the degree of FOXP3 detection and the intensity of H&E staining of the monodermal cellular infiltrate between two independent groups.
The age of the patients ranged from 22 to 59 years, with a median age of 35 years, whereas the age range of the control group was from 22 to 52 years, with a median age of 34.5 years. In terms of the sex distribution, in the patient group, there were 17 men and eight women, whereas in the control group, there were seven men and three women.
In the psoriasis vulgaris group, the PASI score ranged from5 to 40, with a mean score of 12.6, whereas in the erythrodermic psoriasis group, the PASI score ranged from 60 to 70, with a mean score of 65.75.
H&E staining and FOXP3 immunostaining of the psoriatic dermal cellular infiltration and normal skin
In skin biopsy samples taken from the normal skin, few cellular infiltrates composed of mononuclear cells, as assessed by H&E conventional staining, were detected. The mononuclear cells were positive to CD3, CD8, and CD4 antibodies. The percentage of FOXP3+ cells among the dermal mononuclear cells was about 10–20% (Fig. 1).
A significant increase in the intensity of staining of the mononuclear cellular infiltrate was detected on H&E staining in the psoriatic dermis as compared with the normal dermis (U=19.50, P<0.001). The degree of staining ranged from 0 to 1+ in the normal dermis and from 1+ to 2+ in more than half of the psoriatic dermis (Table 1).
Detection of the FOXP3+ cell fraction was significantly higher in the psoriatic dermis than in the normal dermis (U=35.00, P=0.001). It ranged from negative to +1 among controls and from +1 to +3 among most (92%) of the psoriatic patients (Table 1).
Intensity of staining of the mononuclear dermal cellular infiltrate on H&E staining and FOXP3+ cell detection in skin lesions of psoriasis vulgaris and erythrodermic psoriasis
H&E staining in patients with localized and generalized psoriasis vulgaris showed that intensity of staining in the dermal mononuclear cellular infiltrate ranged from mild to severe. In the erythrodermic psoriasis patients, the staining intensity was mild in most of the patients.
The type of psoriasis significantly affected the intensity of staining of the mononuclear infiltrate on H&E staining (U=32.50, P=0.037). Patients with psoriasis vulgaris showed significantly higher levels of mononuclear inflammatory cellular infiltrate than patients with erythrodermic psoriasis.
However, when FOXP3 staining was used, no significant difference was noted in the intensity of staining between the psoriasis vulgaris and erythrodermic psoriasis groups. Yet, it was noted that 29.4% of patients in the psoriasis vulgaris group scored 3+ for FOXP3 compared with 0% in the erythrodermic psoriasis group (Table 2).
In skin lesions of patients with psoriasis vulgaris, FOXP3+ cells were mainly observed in the papillary dermis, consisting of about 20–40% of the mononuclear cellular infiltrate (Fig. 2).
In skin lesions from patients with erythrodermic psoriasis, the percentage of FOXP3+ cells decreased to less than 5–10% of T-cell infiltrates of the papillary dermal mononuclear infiltrates (Fig. 3).
Degree of FOXP3 detection was either 1+ (n=6, 75%) or 2+ (n=2, 25%) among patients with erythrodermic psoriasis and showed no particular distribution among patients with psoriasis vulgaris. Statistically, the degree of detection of FOXP3 was not associated with the severity/chronicity of psoriasis (Table 2).
Correlation between the different parameters studied in the psoriasis vulgaris group versus the erythrodermic psoriasis group
There was a significant strong positive correlation between FOXP3 and the intensity of staining of the mononuclear cellular infiltrate on H&E staining (r=0.781, P<0.001) (Table 3; Fig. 4). PASI was not significantly correlated with the intensity of H&E staining of the mononuclear cellular infiltrate (P=0.369).
PASI was strongly correlated with FOXP3+ cells in the erythrodermic psoriasis group (r=0.765, P=0.027) but not in the psoriasis vulgaris group (P=0.675; Table 3; Fig. 5).
This could be attributed to the fact that the strong direct linear correlation that exists between PASI and the percentage FOXP3+cells is distorted because a number of patients from the vulgaris group scored low in PASI and had a high percentage of FOXP3+ cells at the same time (Fig. 5).
Image cytometric analysis
Image cytometric analysis was carried out for all patient specimens as a semiquantitative tool for objective analysis of FOXP3 intensity scoring. The results of the image analysis were in agreement with those from FOXP3 immunohistochemistry intensity scoring of the specimens.
A representative sample is presented in Fig. 6, showing the evaluation of IHC results for FOXP3 by image analysis (Fig. 7).
CD4+CD25+ Tregs have been suggested to be involved in the pathogenesis of some autoimmune diseases (Gao et al., 2010).
In this study, histopathological examination of skin biopsies from patients with psoriasis and those with normal skin indicated a significant increase in the intensity of H&E staining of the dermal mononuclear cellular infiltrate in the psoriatic dermis compared with the normal dermis (U=19.50, P<0.001).
In the present study, immunohistochemical staining using FOXP3+ monoclonal antibodies was carried out in biopsies from 25 psoriatic patients versus 10 normal controls.
Detection of the FOXP3+ cell fraction was significantly higher in the psoriatic dermis than in the normal dermis. Whereas it ranged from negative to 1+ among controls, it ranged from 1+ to 3+ among most (92%) of the patients.
In agreement with our results, Yan et al. (2010) found that in psoriatic skin lesions, FOXP3+ cells were located predominantly in the papillary and upper reticular layers of the dermis and minimally in the epidermis, whereas in peripheral normal-appearing skin, as in normal controls, FOXP3+ Tregs were scant.
Moreover, Zhang et al. (2010) found that FOXP3+ dermal lymphocytes were more significantly expressed in psoriatic patients than in normal controls.
Patients with psoriasis vulgaris were found to show significantly more intense H&E staining of the dermal mononuclear inflammatory cellular infiltrate compared with patients with erythrodermic psoriasis. When FOXP3 immune staining was performed, no significant difference could be found in the intensity of immune staining between both groups. However, it was observed that 29.4% of patients with psoriasis vulgaris scored 3+ for FOXP3 immune staining of the dermal mononuclear inflammatory cellular infiltrate compared with 0% of erythrodermic psoriasis patients. This last finding is in agreement with the findings of Yan et al. (2010), Chen et al. (2008), and Yun et al. (2010).
Yan et al. (2010) have shown that inflammation can lead to the loss of regulatory function of Tregs and a shift in the effector T-cell balance of Tregs. They hypothesized that the function of FOXP3+ Tregs accumulated in psoriatic lesions might be impaired by proinflammatory cytokines released during the development of disease, and that the differential expression of FOXP3+ Tregs in plaque versus guttate psoriasis may mirror their different causes and immunopathogenesis (Yan et al., 2010).
Chen et al. (2008)) reported that psoriatic lesions in stable and regressive stages contained Tri-Treg in the same frequency range as that in normal human skin, whereas in the progressive stage, the ratio of Tri-Treg versus Teff was much lower compared with that in normal human skin in stable and regressive stages. The reduced ratios in the progressive stage, leading to infiltration of a large quantity of ‘unrestrained’ T cells into the upper dermis, may be responsible for disease progression. The authors concluded that in psoriatic lesions, especially those in the progressive stage, Tregs are lower in number, which may be the reason why they cannot restrain the local ongoing activation of Teff (Chen et al., 2008).
The results of the study carried out by Yun et al. (2010) indicate that the percentage of FOXP3+ Tregs among T lymphocytes increased in the skin lesions of patients with chronic plaque-type psoriasis, but the percentage decreased in the skin lesions of patients with psoriasis with an acute course. On the basis of this finding, the authors suggested that FOXP3+ Tregs play a key role in acute exacerbation when the balance of immunosuppression and immune activation is just about to shift, but not in stable maintenance. It could also be inferred that the increase in Tregs in chronic plaque-type psoriasis might be compensatory to the immune activation, and the consequential immunosuppression might contribute toward the immune balance throughout the course of the disease (Yun et al., 2010).
Zhang et al. (2010) observed significant increases in FOXP3+ lymphocytes among psoriatic patients according to disease severity. These phenomena were accompanied by increases in PASI scores. Hence, infiltration of skin tissue lesions by FOXP3+ lymphocytes was correlated with PASI scores (r=0.867, P<0.001). The authors, whose results were in contrast to those of our study as well as the studies carried out by Yan and colleagues and Chen and colleagues, suggested that differences in detection methodology, the number of enrolled patients and clinical stages, as well as clinical therapy may be the reasons for these inconsistencies (Zhang et al., 2010).
In our study, PASIwas strongly correlated with the percentage of FOXP3+ cells in the erythrodermic psoriasis group but not in the psoriasis vulgaris group.
Yan et al. (2010) reported that the number of FOXP3+ cells was positively correlated with the PASI score of skin lesions (P<0.0001).
In the study carried out by Yun et al. (2010), neither the extent nor the severity of the disease was related to the levels of FOXP3+ Tregs. Rather, the levels of FOXP3+ Tregs were reduced in the lesional skin of acute-onset psoriasis patients.
Zhang et al. (2010) found that the number of FOXP3 cells was positively correlated with the PASI scores of psoriasis patients.
Hence, it could be concluded, that in terms of the correlation of FOXP3 with the PASI score, some studies found the presence of such correlations, whereas others did not. This could be attributed to the difference in the number of patients recruited and the difference in the PASI scores in each of these studies.
We assumed that erythrodermic psoriasis (similar to guttate psoriasis in the study carried out by Yan et al., 2010) is a form of acute psoriasis, in which the level of effector Th17 increases markedly to defend external triggers, whereas the level of FOXP3 Tregs is decreased. However, plaque psoriasis represents a chronic stage of psoriasis in which repeated inflammatory stimuli result in feedback upregulation of FOXP3 Tregs.
Multiple mechanisms have been proposed to explain how Treg cells inhibit effector cells but none can completely explain the observed effects in toto. Proposed mechanisms to explain the suppressive activity of Treg cells include the generation of inhibitory cytokines, induction of death of effector cells by cytokine deprivation or cytolysis, local metabolic perturbation of target cells mediated by changes in extracellular nucleotide/nucleoside fluxes with alterations in intracellular signaling molecules such as cyclic AMP, and finally inhibition of dendritic cell functions. A better understanding of how Treg cells operate at the molecular level could result in novel and safer therapeutic approaches for immune-mediated diseases (Shalev et al., 2011).
Computer-assisted image analysis for FOXP3 staining intensity detection in psoriatic patients versus controls was used because of its major advantage of avoiding interobserver variability in interpretation of subtle antigen-level changes (Hatanaka et al., 2001).
What does this study add?
Higher staining intensity of FOXP3 was noted in patients with psoriasis vulgaris compared with patients with erythrodermic psoriasis.
The FOXP3 level is correlated with the intensity of H&E staining of the mononuclear dermal cellular infiltrate in patients with psoriasis vulgaris but not in those with erythrodermic psoriasis.
FOXP3 intensity scoring was positively and significantly correlated with PASI scoring in patients with erythrodermic psoriasis, whereas in patients with psoriasis vulgaris, this positive correlation did not reach significance.
To the best of our knowledge, this is the first study comparing the FOXP3 staining intensity between patients with psoriasis vulgaris and those with erythrodermic psoriasis (apart from the study by Yun and colleagues, which included only one patient with erythrodermic psoriasis; Chen et al., 2008).
Conflicts of interest
There are no conflicts of interest.
Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al..The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.Nat Genet2001;27:20–21.
Bettelli E, Oukka M, Kuchroo VK.T H-17 cells in the circle of immunity and autoimmunity.Nat Immunol2007;8:345–350.
Buckner JH.Mechanisms of impaired regulation by CD4+ CD25+ FOXP3+ regulatory T cells in human autoimmune diseases.Nat Rev Immunol2010;10:849–859.
Chen L, Shen Z, Wang G, Fan P, Liu Y.Dynamic frequency of CD4+CD25+Foxp3+ Treg cells in Psoriasis vulgaris.J Dermatol Sci2008;51:200–203.
Christophers E.Psoriasis – epidemiology and clinical spectrum.Clin Exp Dermatol2001;26:314–320.
Fujimura T, Okuyama R, Ito Y, Aiba S.Profiles of FOXP3+ regulatory T cells in eczematous dermatitis, psoriasis vulgaris and mycosis fungoides.Br J Dermatol2008;158:1256–1263.
Gao L, Li K, Li F, Li H, Liu L, Wang L, et al..Polymorphisms in the FOXP3 gene in Han Chinese psoriasis patients.J Dermatol Sci2010;57:51–56.
Halabi-Tawil M, Ruemmele FM, Fraitag S, Rieux-Laucat F, Neven B, Brousse N, et al..Cutaneous manifestations of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.Br J Dermatol2009;160:645–651.
Harari M, Shani J, Hristakieva E, Stanimirovic A, Seidl W, Burdo A.Clinical evaluation of a more rapid and sensitive Psoriasis Assessment Severity Score (PASS), and its comparison with the classic method of Psoriasis Area and Severity Index (PASI), before and after climatotherapy at the Dead-Sea.Int J Dermatol2000;39:913–918.
Hatanaka Y, Hashizume K, Kamihara Y, Itoh H, Tsuda H, Yoshiyuki Osamura R, Tani Y.Quantitative immunohistochemical evaluation of HER2/neu expression with HercepTest™ in breast carcinoma by image analysis.Pathol Int2001;51:33–36.
Karai LJ, Bergfeld WF.Recent advances in T-cell regulation relevant to inflammatory dermatopathology: perspective in dermatopathology.J Cutan Pathol2009;36:721–728.
Lowes MA, Kikuchi T, Fuentes-Duculan J, Cardinale I, Zaba LC, Haider AS, et al..Psoriasis vulgaris lesions contain discrete populations of Th1 and Th17 T cells.J Invest Dermatol2008;128:1207–1211.
Nieves DS, Phipps RP, Pollock SJ, Ochs HD, Zhu Q, Scott GA, et al..Dermatologic and immunologic findings in the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.Arch Dermatol2004;140:466–472.
Quaglino P, Ortoncelli M, Comessatti A, Ponti R, Novelli M, Bergallo M, et al..Circulating CD4+CD25FOXP3+ T cells are up-regulated by biological therapies and correlate with the clinical response in psoriasis patients.Dermatology2009;219:250–258.
Shalev I, Schmelzle M, Robson SC, Levy G.Making sense of regulatory T cell suppressive function.Semin Immunol2011;23:282–292.
Sugiyama H, Gyulai R, Toichi E, Garaczi E, Shimada S, Stevens SR, et al..Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: Mechanism underlying unrestrained pathogenic effector T cell proliferation.J Immunol2005;174:164–173.
Valencia X, Yarboro C, Illei G, Lipsky PE.Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus.J Immunol2007;178:2579–2588.
Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA.Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis.J Exp Med2004;199:971–979.
Weinstein GD, Frost P.Abnormal cell proliferation in psoriasis.J Invest Dermatol1968;50:254–259.
Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova J-L, Buist N, et al..X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy.Nat Genet2001;27:18–20.
Yan K-X, Fang X, Han L, Zhang Z-H, Kang K-F, Zheng Z-Z, Huang Q.Foxp3+ regulatory T cells and related cytokines differentially expressed in plaque vs. guttate psoriasis vulgaris.Br J Dermatol2010;163:48–56.
Yun W-J, Lee D-W, Chang S-E, Yoon G-S, Huh J-R, Won C-H, et al..Role of CD4+CD25 high+FOXP3+regulatory T cells in psoriasis.Ann Dermatol2010;22:397–403.
Zhang L, Yang X-Q, Cheng J, Hui R-S, Gao T-W.Increased Th17 cells are accompanied by FoxP3(+) Treg cell accumulation and correlated with psoriasis disease severity.Clin Immunol2010;135:108–117.